a) Field of the Invention
The invention is directed to a method for depth discrimination in optically imaging systems. In particular, it is applicable in light microscopy for achieving an improvement in image quality in the examination of three-dimensionally extending objects.
b) Description of the Related Art
A problem arising in conventional light microscopy when examining three-dimensionally extending objects, i.e., objects whose extension along the optical axis is greater than the depth of focus of the objectives that are used, consists in that the in-focus image from the region of focus is superposed with extra-focal image components which are accordingly imaged out of focus.
In order to overcome this problem, it is known to use confocal imaging in which the light originating from the area outside the region of focus is excluded by means of a pinhole and therefore does not contribute to the image. In this way, an optical section is formed. This confocal point-type imaging requires that the object is scanned in the image plane in order to obtain an image. This scanning can be carried out either by means of scanners in laser scanning microscopes or by means of Nipkow disks.
By recording a plurality of optical section images in different focus positions, a z-stack can be obtained so that the object may be displayed three-dimensionally.
Another method for generating optical sections is to use structured illumination. This was first stated by Meir Ben-Levy and Eyal Pelac in WO 97/6509. This method was improved and expanded as described by Tony Wilson et al. in WO 98/45745 and by Volker Gerstner et al. in WO 02/12945. The disclosure of these three publications is expressly referenced herein.
In WO 97/6509, the object is illuminated by a periodic structure (sine grating or rectangular grating) and an image of the object is recorded by a camera, digitized and stored in a storage. The periodic structure is subsequently displaced within the image plane in such a way that the phase position of the structure is changed and an image is again recorded and stored. This process (displacement, image recording, storage) can be repeated many times. A section image is then generated by calculating from the existing images. The indicated mathematical formulation is a Fourier expansion, which leads to a complicated formula apparatus.
WO 98/45745 shows a simpler formula for the section images which can be derived by simplifying the formula from WO 97/6509 in case of equal phase displacements between the individual recordings.
Realizing the physical boundary conditions required for the application of the indicated method proves very difficult in practice. For example, variation in lamp brightness between the different recordings leads to stripe artifacts in the generated section images. With fluorescing objects, additional problems occur due to the time-dependent fading of the fluorescent dyes, which likewise results in errors. The necessary constancy of the individual phase displacement steps cannot be maintained in practice.
Therefore, it was suggested in WO 02/12945 to compensate for the influence of lamp brightness that varies over time by coupling out part of the light serving to illuminate the object, registering the intensity and subsequently scaling the individual recordings. In order to take into account unequal phase displacement steps, a system of equations (Equation 22, op. cit.) is indicated. To compensate for fading or bleaching, instead of the necessary minimum of three recordings per section image, it is suggested that six recordings are registered in the sequence 1-2-3-3-2-1, two section images are calculated (from 1-2-3 and 3-2-1) and the average is determined therefrom.
A considerable expenditure on instrumentation is required in order to realize these suggestions. Further, the recording of additional images prolongs the required recording time and therefore also increases aging of the sample by illumination with the fluorescence excitation light.